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MyD88 Mediates Instructive Signaling in Dendritic Cells and Protective 1
Inflammatory Response during Rickettsial Infection 2
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Running title: MyD88 is essential for immune control of Rickettsia 5
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Jeremy Bechelli, Claire Smalley, Xuemei Zhao, Barbara Judy, Patricia Valdes, David H. 8
Walker, Rong Fang* 9
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Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, 11
TX USA; 12
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Keywords: MyD88, Rickettsia, immunity, dendritic cells, inflammation, major 14
histocompatibility complex 15
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*Corresponding Author: 17
Rong Fang, M.D. Ph.D, Assistant Professor, Department of Pathology, 18
301 University Blvd., Galveston, Texas 77555-0609, TEL (409) 747-2403, FAX (409) 19
747-2455, E-MAIL: [email protected] 20
21
Conflict of Interest Statement: The authors declare that there is no conflict of interest. 22
23
IAI Accepted Manuscript Posted Online 11 January 2016Infect. Immun. doi:10.1128/IAI.01361-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Abstract (242 words) 24
Spotted fever group rickettsiae cause potentially life-threatening infections throughout 25
the world. Several members of Toll-like receptor (TLR) family are involved in host 26
response to rickettsiae, yet the mechanisms by which these TLRs mediate host 27
immunity remain incompletely understood. In the present study, we found that host 28
susceptibility of MyD88-/- mice to infection with R. conorii or R. australis was significantly 29
greater than wild type (WT) mice, in association with severely impaired bacterial 30
clearance in vivo. R. australis-infected MyD88-/- mice showed significantly lower 31
expression levels of IFN-γ, IL-6 and IL-1β, accompanied by significantly fewer 32
inflammatory infiltrates of macrophages and neutrophils in infected tissues compared to 33
WT mice. The serum levels of IFN-γ, IL-12, IL-6 and G-CSF were significantly reduced 34
while MCP-1, MIP-1α and RANTES were significantly increased in infected MyD88-/- 35
mice compared to WT mice. Strikingly, R. australis infection was incapable of promoting 36
increased expression of MHC-IIhigh and production of IL-12p40 in MyD88-/- bone 37
marrow-derived dendritic cells (BMDCs) compared with WT BMDCs, although co-38
stimulatory molecules were upregulated in both types of BMDCs. Furthermore, the 39
secretion levels of IL-1β by Rickettsia-infected BMDCs and in the serum of infected 40
mice were significantly reduced in MyD88-/- mice compared to WT controls, suggesting 41
that in vitro and in vivo production of IL-1β is MyD88-dependent. Taken together, our 42
results suggest that MyD88 signaling mediates instructive signals in DCs and secretion 43
of IL-1β and type 1 immune cytokines, which may account for the protective 44
inflammatory response during rickettsial infection. 45
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Introduction 48
Rickettsiae are obligately intracellular bacteria that cause potentially life-threatening 49
diseases worldwide, with fatality rates as high as 30% if not treated promptly (1, 2). A 50
U.S. Food and Drug Administration-approved vaccine against rickettsial infections is not 51
available. Our previous studies employing mouse models have identified the critical role 52
of IFN-γ and cytotoxic CD8+ T cells in host clearance of rickettsiae in vivo (2). Increased 53
inflammatory responses, such as serum levels of IFN-γ, TNF-α and IL-6, have been 54
reported in both human mild and severe rickettsioses (3, 4). However, how rickettsiae 55
initiate these inflammatory immune responses in vivo and the contribution of innate 56
immune elements to host protection still remain elusive. 57
58
The initial sensing of infection is mediated by innate pattern recognition receptors 59
(PRRs), which mainly include TLRs and NOD-like receptors (5). Upon activation by 60
microbes, TLRs transduce signals mostly via an adaptor molecule, MyD88 [6]. In 61
addition, activation of TLRs also recruits other adaptor proteins including TIR domain-62
containing adaptor-inducing IFN-β (TRIF) and TRIF-related adaptor molecule (TRAM) 63
(7). These MyD88-dependent and -independent pathways activate the transcription 64
factor NF-κB and the induction of pro-inflammatory cytokines (6-9). MyD88 controls 65
downstream signaling of most TLRs, except for TLR3 and in part TLR4, as well as the 66
IL-1R family (10). Recently, TLR activation by microbial molecules was reported to be 67
essential for upregulation of pro-IL-1β (11). We and others have shown that TLRs are 68
involved in host responses to rickettsial infection. Mice naturally defective in TLR4 69
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signaling (C3H/HeJ) succumb to an inoculum of R. conorii that is sublethal for TLR4-70
competent mice (12). TLR2 has been reported to facilitate cell activation in response to 71
R. akari, the causative agent of rickettsialpox (13). Pre-treatment with CpG-B, a TLR9 72
agonist, protects mice against an ordinarily lethal dose of R. australis, suggesting TLR9 73
is a potential target for vaccine candidates (14). However, the adaptor molecules 74
mediating TLR signaling during rickettsial infection have never been investigated. This 75
problem hampers better understanding of the innate recognition of rickettsiae, which is 76
critical for development of effective therapeutic interventions and vaccine candidates. 77
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Dendritic cells (DCs) are the critical sentinel in antimicrobial immune responses. TLR 79
signaling is one of the most important mediators for DC maturation which results in 80
priming naïve T cells and links the innate and adaptive immune systems (15). Our 81
previous studies have shown that high host susceptibility to R. conorii is associated with 82
an altered DC maturation profile and low levels of IL-12p40 secretion by infected 83
BMDCs, which result in delayed CD4 T cell activation and suppressed Th1 cell 84
polarization (16). Adoptive transfer of rickettsiae-stimulated DCs rescues the host from 85
a lethal challenge with R. conorii (17). In the present study, we hypothesized that 86
MyD88 is a critical molecule mediating host protective inflammation and immunity 87
against fatal murine rickettsioses via signaling TLR activation in innate immune cells 88
such as DCs. 89
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Materials and Methods 95
Rickettsia and mice 96
R. australis (Cutlack strain) and R. conorii (Malish 7 strain) used for animal inoculation 97
were propagated in pathogen-free embryonated chicken eggs (18). For in vitro DCs 98
infection, R. australis was cultivated in Vero cells and purified as previously described 99
with modifications (16). Briefly, infected cells were placed on the top of 32%, 26% and 100
20% OptiPrep Density Gradient medium in 6 × SPG buffer (218 mM sucrose, 3.76 mM 101
KH2PO4, 7.1 mM K2HPO4, 4.9 mM potassium glutamate) bed (Sigma-Aldrich, St. Louis, 102
MO) after sonication. All rickettsial stocks were quantified by plaque assay and stored at 103
-80°C until use. WT C57BL/6 (B6) mice and TLR2-/- mice were purchased from Jackson 104
Laboratories (Bar Harbor, Maine). MyD88-/- mice were kindly provided by Dr. Tina Wang 105
at University of Texas Medical Branch (UTMB) and Dr. Shizuo Akira of Osaka University 106
(Japan). For in vivo experiments, mice were inoculated intravenously (i.v.) through the 107
tail vein with R. australis or R. conorii at the doses indicated. After infection, mice were 108
monitored daily for signs of illness until day 20 post infection (p.i.). To determine the 109
bacterial loads in tissues and immune response in vivo, mice were sacrificed on days 1 110
and 4 p.i.. All the experiments described in this study were performed in a certified 111
biosafety level 3 (BSL3) laboratory at UTMB. All mice were maintained and manipulated 112
in an animal biosafety level-3 (ABSL3) facility at UTMB. The UTMB Animal Care and 113
Use Committee approved all experiments and procedures, and experiments in mice 114
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were performed according to the guidelines of the Guide for the Care and Use of 115
Laboratory Animals. 116
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Quantitative detection of rickettsiae and inflammatory cytokines in infected 118
tissues 119
Mouse lung, liver and spleen were collected in RNAlater (Thermo Fisher Scientific, 120
Waltham, MA). Rickettsial loads in the mouse tissues were quantified using quantitative 121
PCR following DNA extraction as described in our previous studies (18). Total RNA was 122
prepared using Qiagen RNeasy Mini kit (Valencia, CA) following the manufacturer’s 123
recommendations. Reverse transcription (RT) was performed using isolated and 124
DNase-treated RNA with Bio-Rad iScript cDNA synthesis kit (Hercules, CA). The 125
resulting cDNAs were used as template for quantitative reverse transcription–126
polymerase chain reaction (RT-PCR). Gene expression of individual genes was 127
determined using SYBR Green PCR Master Mix on an iCycler IQ (Bio-Rad, Hercules, 128
CA). The ∆∆Ct method was used as described previously with modifications on 129
housekeeping gene (19). Quantification results were expressed as the mRNA relative 130
ratio (2-∆∆Ct) normalized to the amount of β-actin housekeeping gene. Analyzed genes 131
and primers are shown in Table 1 (20-22). 132
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In vivo systemic cytokine release evaluation 134
Serum samples were collected from infected mice on days 1 and 4 p.i.. Uninfected 135
mouse serum served as controls. The analysis of 14 cytokines and chemokines was 136
performed on serum samples using a magnetic bead-based multiplex immunoassay 137
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(Bio-Plex; BIO-RAD Laboratories, Hercules, CA) following the manufacturer’s 138
instructions. These cytokines and chemokines included IFN-γ, TNF-α, IL-10, IL-4, IL-5, 139
IL-6, IL-12p40, IL-12p70, IL-13, IL-17A, granulocyte-colony stimulating factor (G-CSF), 140
monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α 141
(MIP-1α), and regulated on activation, normal T cell expressed and secreted (RANTES). 142
The plates were read at 450 nm, and the absorbances were transformed to pg/ml using 143
calibration curves prepared with cytokine standards included in the kit. 144
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Histopathological and immunohistochemical analyses 146
Formalin-fixed, hematoxylin and eosin (H&E)-stained uninfected and infected tissue 147
sections were evaluated by a pathologist by both low- (X 10) and high-magnification (X 148
40) microscopy. Images were taken using an Olympus BX41 photomicroscope 149
(Olympus America Inc., Center Valley, PA). The frequency and size of inflammatory 150
infiltrates in the liver were measured by Image J as described previously (23). Antigen 151
unmasking was performed by treatment with sodium citrate buffer at pH 6, and tissue 152
sections were stained for neutrophils (Abcam, clone NIMP-R14) and macrophages 153
(Santa Cruz, clone sc-71088). Biotinylated secondary antibodies (Vector Labs) were 154
diluted in Dako antibody diluent (Dako, #S3022). The tertiary reagents, 155
streptavidin/alkaline phosphatase, were diluted in the same Dako diluent, as were 156
primary antibodies. 3,3'-diaminobenzidine (DAB) was used as the chromogen and 157
hematoxylin as the counterstain. All washes were with TBS Tween-20 (Sigma). 158
Sections were dehydrated before the synthetic glass coverslips were mounted with 159
permount mounting medium. All the sections were photographed with an Olympus 160
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DP71 camera (Olympus, Center Valley, PA, USA) attached to an Olympus Ix71 Inverted 161
Microscope (Olympus, Tokyo, Japan) utilizing × 20 objectives. We counted the stained 162
cells using previously described Image J-based method (24). 163
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Generation of bone marrow-derived dendritic cells (BMDCs) 166
Generation of primary BMDCs from WT mice and MyD88-/- mice was performed as 167
previously described (16). Briefly, a single-cell suspension from bone marrow was 168
prepared from the mouse femurs and adjusted to 106 cells per 10 ml of complete 169
Iscove's modification of Dulbecco's modified Eagle's medium containing 10% low 170
endotoxin fetal bovine serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol). DC 171
culture medium was supplemented with 20 ng/ml recombinant granulocyte-macrophage 172
colony-stimulating factor (GM-CSF; eBioscience, San Diego, CA). At day 3, 6 ml of 173
fresh GM-CSF-containing medium was added, and 10 ml of the culture medium was 174
replaced with fresh GM-CSF-containing medium at day 6. On day 8, cells were 175
analyzed by flow cytometry and used if they contained ≥70% CD11c+ cells. 176
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Flow cytometry 178
BMDCs were incubated for 24 h with medium, R. australis or 500 ng/ml LPS (Sigma, 179
L4391). Cells were first stained with LIVE/DEAD® fixable staining kit (Life Technologies, 180
Grand Island, NY) for 30 minutes. Fc receptors were blocked with anti-CD16/32 (Fc 181
Block; BD Biosciences). Cells were then stained with fluorophore-labeled monoclonal 182
antibodies for evaluation of the maturation status. Antibodies including fluorescein 183
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(FITC)-coupled anti-CD80 (clone 16-10A1), and FITC coupled anti-MHC II (clone 2G9) 184
were purchased from BD Bioscience (San Jose, CA), and antibodies allophycocyanin 185
(APC) or PerCP-Cy5.5 coupled anti-CD11c (clone N418), APC-conjugated anti-CD40 186
(clone 1C10), and FITC coupled anti-CD86 (clone GL1) were purchased from 187
eBioscience (San Diego, CA). Cells were fixed in 1% paraformaldehyde prior to analysis 188
by a FACSCalibur flow cytometer (Becton Dickinson). Data were analyzed using FlowJo 189
software (Tree Star, Inc.). 190
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Cytokine enzyme-linked immunosorbent assay (ELISA) 192
Supernatants of BMDCs or sera were collected and either filtered or treated with 0.9% 193
sodium azide to eliminate live R. australis. Cytokine concentrations were measured by 194
using IL-12p40 Quantikine ELISA kit (R&D Systems, Minneapolis, MN) or IL-1β ELISA 195
kit (eBioscience, San Diego, CA). Absorbance was measured using VersaMax 196
microplate reader (Molecular Devices, Sunnyvale, CA). The limits of detection of the 197
ELISA for cytokine measurements were as follows: IL-1β, 4.0 pg/ml; IL-12p40, 2.0 198
pg/ml. 199
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Statistical analyses 201
For comparison of multiple experimental groups, the one-way analysis of variance 202
(ANOVA) with Bonferroni's procedure was used. Two-group comparison was conducted 203
using either Student’s t-test or Welch's t-test depending on whether the variance 204
between two groups was significantly different. To determine whether the difference in 205
survival between different mouse groups was significant, data were analyzed by the 206
Gehan-Breslow-Wilcoxon Test. All the statistical analyses were performed using 207
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GraphPad Prism software version 5.01. P values of 0.05 or less were the threshold for 208
statistical significance. 209
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Results 214
MyD88 mediates host clearance of R. conorii in vivo 215
To investigate whether MyD88 is involved in host immunity against rickettsiae, we first 216
compared the survival of WT and MyD88-/- mice in response to R. conorii infection. In 217
line with the previous studies, WT mice were resistant to different doses of R. conorii 218
(data not shown) (25). In contrast, 80% of MyD88-/- mice died on day 1 p.i. and 20% of 219
MyD88-/- mice died on day 6 p.i. after intravenous inoculation with R. conorii at a dose of 220
5.7 × 105 plaque forming units (PFUs) (Fig. 1A). These results suggest that MyD88-/- 221
mice died of mouse toxicity resulting from a very high dose of R. conorii (26, 27). In 222
response to a lower dose of R. conorii (3 ×105 PFUs), all MyD88-/- mice survived (Fig. 223
1A), indicating dose-dependent mortality of MyD88-/- mice to rickettsial infection. The 224
concentration of Rickettsia in the liver was dramatically greater in MyD88-/- mice 225
compared to that in WT mice on day 2 p.i. (Fig. 1B). To investigate the involvement of 226
TLR2 in the MyD88-dependent host protective immune response against R. conorii, we 227
inoculated TLR2-/- and WT mice with the same dose of Rickettsia, and we did not find 228
any significant difference in host survival (data not shown). 229
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MyD88 plays an essential role in host defense against R. australis 231
To further determine the contribution of MyD88 to host immunity against rickettsiae, we 232
challenged MyD88-/- mice and WT mice with the same dose of R. australis. R. australis 233
establishes a dose-dependent lethal infection in WT B6 mice (28), which is the murine 234
background on which most of the gene knockout mice have been developed. When 235
challenged with R. australis at a dose of 2.4 × 106 PFUs per mouse, all WT mice 236
survived while all MyD88-/- mice died on days 5 and 6 p.i. (Fig. 2A). To determine the 237
growth kinetics of rickettsiae, we inoculated WT and MyD88-/- mice i.v. with R. australis 238
at a dose of 8 × 105 PFU per mouse and measured the bacterial burden in the liver on 239
days 1 and 4 p.i.. We observed dramatic and significantly greater rickettsial loads in the 240
liver of MyD88-/- mice compared to WT mice on day 4 p.i., but not on day 1 p.i. (Fig. 2B). 241
A significantly greater rickettsial burden was also present in spleen and lung of MyD88-/- 242
mice compared to WT mice on day 4 p.i. (Fig. 2C) (approximately 3-log higher in each 243
tissue examined). These data illustrate that the decreased host survival of MyD88-/- 244
mice in response to R. australis parallels severely impaired control of bacterial growth in 245
vivo. 246
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R. australis induces MyD88-dependent and -independent inflammatory cytokines 248
in specific infection sites 249
To investigate the cytokines involved in MyD88-mediated host immune protection 250
against rickettsiae, we assayed the pro-inflammatory and anti-inflammatory cytokines at 251
the transcriptional level by RT-PCR in the specific infection sites during R. australis 252
infection in vivo. On day 1 p.i., most of the cytokines were not significantly altered 253
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except for significantly lower levels of IFN-γ, IL-6 and IL-1β in lung of MyD88-/- mice 254
compared to WT mice (Fig. 3). These results suggest that intravenous injection of 255
rickettsiae initiated an immediate MyD88-dependent type 1 cytokine response in lung. 256
On day 4 p.i., the expression levels of IFN-γ (lung, liver and spleen), TNF-α (lung and 257
spleen), IL-6 (lung and liver) and IL-1β (lung) were all significantly less in MyD88-/- mice 258
compared to WT mice, indicating MyD88-dependent production of these inflammatory 259
cytokines in the infection sites. These results suggest that IFN‑γ, representing the 260
antimicrobial type 1 immune response, and IL-1β were invoked on day 4 p.i. in a 261
MyD88-dependent manner. Interestingly, the mRNA expression of TNF-α and IL-10 in 262
liver, and IL-6 in spleen was significantly greater in MyD88-/- mice compared to WT mice 263
on day 4 p.i. (Fig. 3), suggesting that these cytokines were initiated by a MyD88-264
independent mechanism or tissue-specific MyD88 signaling. These MyD88-independent 265
cytokines are not likely involved in host protection against rickettsiae. Our previous work 266
has shown the association of IL-10 with the immunosuppression induced by a lethal 267
dose of rickettsial infection (18). IL-6 also mediates the production of T regulatory cells 268
(29). However, it still remains unknown whether IL-6 and IL-10 mediate 269
immunosuppression during murine fatal and severe rickettsioses. Taken together, our 270
data suggest that MyD88 mediates host protection against R. australis in vivo mainly 271
through a type 1 immune response. 272
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R. australis induces circulating MyD88-dependent and -independent 274
cytokines/chemokines 275
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To further investigate the mechanisms by which MyD88 mediates the protective 276
inflammatory responses upon rickettsial infection in vivo, we measured the production 277
levels of cytokines and chemokines in the serum of WT and MyD88-/- mice infected i.v. 278
with R. australis at a dose of 8 × 105 PFU per mouse. In line with the insignificant 279
changes in bacterial concentration in tissues on day 1 p.i. (Fig. 2B), we did not find any 280
significant difference in serum cytokines/ chemokines in WT B6 vs. MyD88-/- mice either 281
before infection or on day 1 p.i. (Fig. 4). On day 4 p.i., the serum levels of IFN-γ, IL-282
12p40, IL-12p70, IL-6 and G-CSF were significantly lower in MyD88-/- mice compared to 283
those in WT mice (Fig. 4), suggesting that MyD88 signaling mediates a systemic 284
inflammatory response oriented towards a type 1 cytokine profile, and IFN-γ is a critical 285
cytokine mediating the protective immunity against rickettsiae in vivo. Serum levels of 286
TNF-α and IL-10 were not significantly altered in MyD88-/- mice vs. WT mice, suggesting 287
that these two cytokines were produced systemically in a MyD88-independent manner. 288
No significant levels of Th2 or Th17 cell cytokines such as IL-4, IL-5, IL-13 and IL-17 289
were detected in these samples (data not shown), suggesting that a Th1 cell response 290
was dominantly induced. Interestingly, the serum levels of MCP-1, MIP-1α and 291
RANTES were significantly greater in MyD88-/- mice compared to WT mice, suggesting 292
that these chemokines are MyD88-independent or a deficiency of MyD88 may indirectly 293
promote production of these chemokines. 294
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MyD88-dependent infiltration of inflammatory cells in infection sites during 296
rickettsial infection in vivo 297
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We next examined the inflammatory cell recruitment following secretion of inflammatory 298
cytokines/chemokines in lung and liver of WT and MyD88-/- mice. Histologic analysis of 299
the H&E stained organ sections showed randomly distributed lobular foci of cellular 300
infiltration consisting of predominantly macrophages, but also lymphocytes and variable 301
numbers of neutrophils in the liver of infected animals (Fig. 5A). The lung sections 302
showed no obviously distinguishing pathological features in infected WT B6 and 303
MyD88-/- mice (Supplementary Fig. 1). Interestingly, we found significantly less 304
frequency and smaller size of inflammatory infiltrations in liver of MyD88-/- mice 305
compared to WT mice on day 4 post infection with R. australis (Figs. 5A and B). 306
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To further characterize the inflammatory infiltration mediated by MyD88, we determined 308
the type and frequency of infiltrated inflammatory cells in the liver and lung on day 4 p.i. 309
by immunohistochemical quantification and analysis. Macrophages and neutrophils 310
were stained with individual specific antibodies. In vivo R. australis infection initiated 311
significantly greater infiltration of macrophages in the liver and neutrophils in the lung in 312
WT B6 mice compared to MyD88-/- mice (Fig. 5C). These results suggest that MyD88-313
dependent expansion or trafficking of macrophages in liver and neutrophils in lung 314
contributed to the host protective immune response against R. australis infection. 315
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MyD88 is required for upregulation of MHC-IIhigh, but not co-stimulatory 317
molecules, on BMDCs as well as for IL-12p40 production following rickettsial 318
infection 319
To further identify the mechanisms by which MyD88 mediates a protective immune 320
response and the recognition of bacteria by innate PRRs during rickettsial infection, we 321
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investigated the maturation of BMDCs of MyD88-/- mice and WT mice upon infection 322
with R. australis. Upon LPS stimulation, the levels of MHC-IIhigh, CD86, CD80, and 323
CD40 in both MyD88-/- and WT mice were greatly enhanced compared to uninfected 324
controls (Fig. 6A). R. australis infection induced full maturation of WT DCs as evidenced 325
by significantly increased percentages of DCs expressing maturation markers compared 326
to uninfected control cells, including MHC-IIhigh (22.2% vs.14.6%), CD86 (19.8% vs. 327
11.9%), CD80 (25.2% vs. 18.8%) and CD40 (15.2% vs. 8.5%). In contrast, R. australis 328
infection failed to increase the frequency of MyD88-/- DCs expressing MHC-IIhigh (23.9%) 329
compared to uninfected controls (23.6%) although co-stimulatory molecules in MyD88-/- 330
BMDCs were similarly enhanced upon infection as WT BMDC (Fig. 6A). The fold 331
increase in expression levels of MHC-IIhigh on infected WT BMDCs (> 1) was 332
significantly higher than those on infected MyD88-/- BMDCs (≤1), suggesting that MHC-333
IIhigh was only significantly upregulated in WT BMDCs but not MyD88-/- BMDCs upon 334
rickettsial infection (Fig. 6B). No significant difference was observed in fold increase in 335
expression levels of co-stimulatory molecules in WT vs. MyD88-/- BMDCs (Fig. 6B). 336
These results suggest that R. australis induced the MyD88-independent upregulation of 337
CD86, CD80, and CD40 in BMDCs while the enhancement of MHC-IIhigh molecule is 338
MyD88-dependent. During maturation, DCs increase their surface expression of MHC-II 339
molecules several fold (30), and proceed to efficient antigen processing and 340
presentation to T cells. Our results suggest that the full maturation of DC driven by 341
Rickettsia is MyD88-dependent. 342
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IL-12 secreted by DCs is critical and instructive for pathogen-driven Th1 cell polarization 344
(29). Uninfected WT and MyD88-/- BMDCs produced the same levels of IL-12p40 (data 345
not shown). Importantly, no significantly increased secretion of IL-12p40 (fold increase 346
<1) was detected in MyD88-/- BMDCs upon rickettsial infection, although infected WT 347
BMDCs produced approximately 1.5 fold greater amount of IL-12p40 compared to 348
uninfected controls (Fig. 6C). In addition, the levels of maturation markers on splenic 349
DCs from WT and MyD88-/- mice are consistent with what we have described in vitro 350
(Supplementary Fig. 2). These results suggest that MyD88 mediates the instructive 351
signals in DCs, which may serve as the key mechanism of host protective inflammatory 352
response to R. australis. 353
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MyD88-dependent IL-1β production in vitro and in vivo 355
IL-1β is a well-documented cytokine mediating the inflammatory response. Recent 356
studies have demonstrated the critical role of inflammasomes in the production of IL-1β 357
as well as the involvement of MyD88 in the induction of pro-IL-1β (11). To further 358
investigate whether IL-1β is one of the downstream effectors mediated by MyD88 during 359
rickettsial infection, we measured the in vitro and in vivo production of IL-1β in WT and 360
MyD88-- mice. We did not find a significant level of IL-1β produced by uninfected 361
BMDCs (data not shown). Upon R. australis infection, WT BMDCs produced a 362
significantly higher level of IL-1β than that in MyD88-/- mice (Fig. 7A), suggesting that 363
MyD88 is required for secretion of IL-1β in these infected cells. In line with the in vitro 364
findings, the levels of IL-1β in the serum of infected MyD88-/- mice were significantly 365
lower compared to WT mice (Fig. 7B), suggesting that IL-1β production is dependent on 366
MyD88 in vivo. Taken together, our results suggest that inflammasome-derived IL-1β is 367
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the critical component of the MyD88-mediated inflammatory response to rickettsial 368
infection as demonstrated both in vitro and in vivo. 369
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Discussion 382
Although the available evidence suggests that multiple TLRs, including TLR2, TLR4 and 383
TL9 (12-14), are involved in host responses to Rickettsia, it is not clear how signals from 384
different TLRs are orchestrated in generating the immune response and their 385
contribution to host resistance against rickettsiae in vivo. In this study, we demonstrated 386
that MyD88 was essential for host resistance to R. australis as evidenced by 387
significantly greater control of rickettsial replication and enhanced host survival in 388
infected WT mice compared to MyD88-/- mice. Our results also suggest that MyD88, 389
which likely serves as the essential adaptor of key TLRs and priming signal for 390
biologically functional IL-1β secretion, was required for sensing rickettsiae and inducing 391
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a protective inflammatory cytokine response as well as cellular infiltrations during 392
infection in vivo with this endothelium-targeting intracellular bacterium. 393
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MyD88 signaling plays distinct roles in host responses against several intracellular 395
pathogens closely related to rickettsiae. MyD88-deficient mice infected with Ehrlichia 396
muris exhibit reduced serum and bone marrow concentrations of IFN-γ, which is 397
required for controlling infection, compared to WT mice (31, 32). Interestingly, while in 398
vivo clearance of Anaplasma phagocytophilum is completely independent of MyD88 399
(33), MyD88−/− mice have less severe histopathological changes than infected controls 400
(34). In contrast, our results illustrated that MyD88 not only mediated host clearance of 401
rickettsiae in vivo, but also contributed to inflammatory cellular infiltrations in vivo during 402
R. australis infection. 403
404
Dendritic cells mediate pathogen-driven T cell polarization through antigen-specific 405
signal presented by MHC class II molecules, costimulatory molecules and polarizing 406
cytokines (35). MyD88-dependent or -independent TLRs in DCs play a critical role in 407
sensing microbial pathogens resulting in DC maturation and subsequent T cell priming. 408
Maturation of DCs stimulated by both Brucella abortus and Mycobacterium tuberculosis 409
requires MyD88 as demonstrated by abrogated upregulation of CD80, CD86 and MHC 410
class II as well as IL-12 production in MyD88-deficient DCs upon infection (9, 36). 411
MyD88-deficient BMDCs infected with respiratory syncytial virus upregulate 412
costimulatory molecules, but do not upregulate class II as efficiently as do WT BMDCs 413
(37), which is very similar to our findings with rickettsial infection. It is known that 414
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MyD88-deficient BMDCs do not mature in response to bacterial DNA, the ligand for 415
TLR9, but undergo full maturation upon intracytoplasmic TLR4 activation by LPS (15). 416
As shown in Fig 6, our results suggest that R. australis activates TLRs other than TLR9, 417
possibly TLR4, to promote MyD88-independent upregulation of co-stimulatory 418
molecules in DCs. However, R. australis activates TLRs other than intracytoplasmic 419
TLR4 to promote MyD88-dependent upregulation of MHC-class II high and IL-12 420
production in DCs, which are both essential for induction of antigen-specific Th1 cell 421
responses. Although Jordan et al. demonstrated the critical role of TLR4 in mediating 422
host protection against R. conorii in vivo, the maturation profile of DCs from mice 423
carrying the TLR4 point mutation is not known (12). It is an attractive hypothesis that 424
rickettsial antigen, which accounts for MyD88-dependent enhancement of MHC-II high on 425
DCs, serves the most potent or essential component of vaccine candidates for 426
controlling of rickettsial infection via type 1 immune responses. Although future 427
investigations are required to reveal whether MyD88 also functions as an adaptor 428
protein downstream of the IL-1R family during rickettsial infection, our current data at 429
least suggest the contribution of MyD88-dependent TLR signaling to innate recognition 430
of rickettsiae in vivo. 431
432
Our study clearly demonstrated the immune mechanisms mediated by MyD88 during in 433
vivo infection with rickettsiae. However, it is worthy to note that the production levels of 434
CC-chemokines (MCP-1, RANTES and MIP-1α) in circulation were MyD88-435
independent. Rickettsia has been shown to induce these CC-chemokines in endothelial 436
cells in vitro (38). Given the severity of rickettsioses in MyD88-/- mice as indicated by 437
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dramatically greater concentrations of rickettsiae in infected tissues, these CC-438
chemokines were likely produced by infected endothelial cells and may play a role in the 439
late stage of pathogenesis of rickettsial diseases. We propose that significantly 440
increased serum levels of MCP-1, RANTES and MIP-1α could be potentially used as 441
biomarkers of human rickettsioses. Of note, on day 4 p.i., although the expression of 442
cytokine transcripts including TNF-α and IL-10 in liver and IL-6 in spleen was 443
significantly upregulated in MyD88-/- mice (Fig. 3), the serum levels of TNF-α, IL-10 and 444
IL-6 were either insignificantly altered or significantly decreased in MyD88-/- mice (Fig. 445
4). These results suggest that the in vivo induction of these pro-inflammatory (TNF-α 446
and IL-6) and anti-inflammatory (IL-10) cytokines by rickettsial infection may involve 447
MyD88-independent or tissue-specific MyD88 signaling. 448
449
Overall, our studies revealed that MyD88 is a key molecule in the immune surveillance 450
system mediating the innate recognition of rickettsiae in vivo. These findings suggest 451
that MyD88-mediated signaling pathways could serve as potential targets in vaccine 452
design and therapeutic interventions during this intracellular bacterial infection. 453
454
Funding information 455
This work was supported by grants AI021242 and AI101413 from the National Institute 456
of Allergy and Infectious Diseases. Jeremy Bechelli is supported by the PT32-AI060549 457
Biodefense Training Grant at the University of Texas Medical Branch. 458
459
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Acknowledgements 460
We thank Kerry Graves at UTMB for his professional assistance with 461
immunohistochemical staining. 462
463
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Figure legends 611
Fig 1 MyD88 mediates host clearance of R. conorii in vivo. A, MyD88-/- mice were 612
inoculated i.v. with different doses of R. conorii as indicated. Host survival was 613
monitored daily until day 10 p.i.. B, WT and MyD88-/- mice were injected i.v. with R. 614
conorii at a dose of 3 × 105 PFUs /mouse. On day 2 p.i., mice were sacrificed and the 615
concentrations of rickettsiae in liver were quantified by real-time PCR. Data represent 616
mean ± SD for n=4 mice for each group. Data represent two independent experiments. 617
*, p<0.01 for significant difference between WT and MyD88-/- mice. 618
619
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Fig 2 MyD88 is essential for host control of R. australis in vivo. A, WT and MyD88-/- 620
mice were inoculated i.v. with R. australis at a dose of 2.4 × 106 PFUs per mouse. Mice 621
were monitored for survival daily until day 10 p.i.. To determine the growth kinetics of R. 622
australis in WT and MyD88-/- mice, mice were inoculated with 8 × 105 PFU of R. 623
australis i.v.. The bacterial burden in liver on days 1 and 4 p.i. (B) and the 624
concentrations of rickettsiae in spleen and lung on day 4 p.i. (C) were determined. *, 625
p<0.01 for a significant difference between WT and MyD88-/- mice. 626
627
Fig 3 In vivo production of MyD88-dependent and -independent inflammatory cytokines 628
upon rickettsial infection. WT and MyD88-/- mice were infected i.v. with R. australis (8 × 629
105 PFU per mouse). On days 1 and 4 p.i., mice were sacrificed and tissues were 630
isolated. Total RNA was extracted from these infected tissues. The transcriptional levels 631
of cytokines including IFN-γ, TNF-α, IL-6, IL-1β and anti-inflammatory cytokine IL-10 632
were determined by RT-PCR as described in Materials and Methods. *, p<0.05 using 633
unpaired t test. 634
635
Fig 4 Systemic production of cytokines/chemokines in infected WT and MyD88-/- mice. 636
Mice were infected with R. australis i.v. and then sacrificed on days 1 and 4 p.i.. Serum 637
levels of cytokines/chemokines including IFN-γ, TNF-α, IL-10, IL-4, IL-5, IL-6, IL-10, IL-638
12p40, IL-12p70, IL-13, IL-17A, G-CSF, MCP-1, MIP-1α and RANTES were assessed 639
by Bioplex assay. Cytokines with insignificantly different levels between infected 640
MyD88-/- and WT mice are not shown here except IL-10 and TNF-α. Results are means 641
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± SD of data from 4-10 mice per group. * Significant difference in MyD88-/- mice vs. WT 642
mice (p<0.05). 643
644
Fig 5 MyD88-dependent inflammatory cellular accumulation in vivo. WT and 645
MyD88 -/- mice were infected i.v. with R. australis (8 × 105 PFU per mouse). On day 4 646
p.i., mice were sacrificed and tissues were isolated and analyzed. A, Histology of liver 647
from infected mice under magnifications 10 X and 40 X. Foci of inflammatory infiltration 648
are shown by arrows. B, The size and frequency of inflammatory lesions were analyzed 649
using Image J (magnification 20 X). C, Infected tissues were further stained with specific 650
antibodies against F4/80 and NIMP-R14 by immunohistochemistry for detection of 651
macrophages and neutrophils, respectively. Frequencies of inflammatory cells in 652
different tissues were compared. Randomly selected 15 microscopic fields from 653
immunohistochemically stained tissues were analyzed for each comparison. These 654
results are representative of two independent experiments (n=8). *, p<0.05 using 655
unpaired t test. 656
657
Fig 6 MyD88-dependent and -independent upregulation of maturation markers on 658
BMDCs after R. australis infection in vitro. BMDCs were isolated from WT and MyD88-/- 659
mice as described in Materials and Methods. Cells were left untreated, stimulated with 660
LPS (500 ng/ml) or infected with R. australis at a multiplicity of infection (MOI) of 5 for 661
24 h. A, Cells were collected and evaluated for maturation status by expression levels of 662
MHC-II, CD86, CD80 and CD40 on gated CD11c+ cells using flow cytometric analysis. 663
B, The frequencies of R. australis-infected cells expressing differential maturation 664
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markers were calculated for fold increase based on comparison with unstimulated 665
controls. C, IL-12p40 secretion driven by rickettsial infection was assayed by ELISA and 666
was calculated for fold-change based on comparison with uninfected controls. These 667
results are representative of three independent experiments. *, p ≤ 0.05. 668
669
Fig 7 MyD88-dependent secretion of IL-1β during rickettsial infection in vitro and in vivo. 670
A, BMDCs isolated from WT and MyD88-/- mice were infected with R. australis (MOI=5) 671
for 24 h. IL-1β secretion by these infected cells was assayed by ELISA. B, WT and 672
MyD88-/- mice were infected i.v. with R. australis (8 × 10^5 PFU per mouse). On day 4 673
p.i., mice were sacrificed, and the levels of IL-1β in sera were evaluated by ELISA. 674
These results are representative of two independent experiments. *, p<0.05. 675
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Table 1. Primer Sequences Used to Quantify mRNA of Genes of Interest
Name and Sequence (5’3’) Reference
β-actin: forward (F), GAT TAC TGC TCT GGC TCC TAG C
reverse (R), GAC TCA TCG TAC TCC TGC TTG C
IL-6: forward (F), CAC AAG TCC GGA GAG GAG AC
reverse (R), CAG AAT TGC CAT TGC ACA AC
IFN-γ: forward (F), GTT ACT GCC ACG GCA CAG TCA TTG
reverse (R), ACC ATC CTT TTG CCA GTT CCT CCA G
IL-10: forward (F), ACC AAC ATC CTG GTG TCT CC
reverse (R), CAT GTC AAA CGT GAG CGA CT
TNF-α: forward (F), GCA AGC TTC GCT CTT CTG TCT ACT
GAA CTT CGG
reverse (R), GCT CTA GAA TGA GAT AGC AAA
TCG GCT GAC GG
IL-1β: forward (F), CGC AGC AGC ACA TCA ACA AGA GC
reverse (R), TGT CCT CAT CCT GGA AGG TCC ACG
20, 21
21, 22
22
22
22
20, 21
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